This invention relates to the development and commercial production of helminths (parasitic worm) antigens.
The use of antigens for diagnostics and vaccines to detect and prevent infectious diseases such as polio, smallpox, diphtheria, tetanus and hoof and mouth diseases has been clearly shown in both humans and animals. Most antigens used as diagnostics and vaccines are derived from cultured infectious organisms. The infectious organisms are grown in vitro or in vivo in animals or tissue cultures. An example is polio antigens which are derived from viruses grown in vitro in monkey kidney cells. The antigens are used as a diagnostic and to vaccinate humans.
If a small amount of attenuated viruses is inoculated in a host, the inoculated viruses which carry specific antigens will induce antibody formation in the host which in the future will recognize and destroy future invading polio viruses. The same polio virus antigens can also be used for the diagnosis of a polio infection. If a host is infected by polio, some specific antibodies will be elicited in the body and the presence of these antibodies will be indicative of an infection. The detection of antibodies can be assayed for their binding to polio antigens with the standard immunofluoroscent, radio-immune, and enzyme-linked, immunoelectrophoresis, hemagglutinin and immunodiffusion assays.
It is not possible, however, to commercially produce antigens for all infectious diseases. One limitation of production has been the inability to cultivate or produce a large amount of infectious organisms in vivo or in vitro from which the antigens are derived. This is especially true for vaccines in which the infectious organisms have complicated life cycles and/or have more than one host. Most parasitic worm diseases that infect dogs, cats, sheep, pigs, horses and humans fall into this category.
An example is the helminths disease of the heartworm which can infect a wide variety of organisms from dogs, cats, seals to humans (infrequently). The parasite, however, generally resides in the dog as its host with the mosquito as its intermediate host. It would be difficult to derive antigens necessary in the manufacture of vaccines or diagnostics since the parasitic worm undergoes several stages of larval development and only one larval stage contains the appropriate antigens. In the heartworm this particular stage, the infectious larvae, resides in the mosquito. Under these conditions, the capture of the intermediate hosts (mosquitos) and the dissection for the infectious larvae is necessary to generate required antigens for the manufacture of a heartworm vaccine. Wong, M. M., Guest, M. F., and Laviopierre, M. J. (1974) Dirofilaria immitis; Fate and Immunogenicity of Irradiated Infective Stage Larvae in Beagles Experimental Parasitology 35, 65-74. Similarly, antigens which are necessary to diagnose heartworm antibodies for infection must be derived from adult heartworms harbored in the heart of an infected dog. Desowitz, R. S. and Una, S. R. (1976), The Detection of Antibodies in Human and Animal Filariasis by Counter-immunoelectrophoresis with Dirofiliaria immitis Antigen. Journal of Helminthology, 50, 53-57, Grieve, R. B., Mika-Johnson, M., Jacobson, R. H., and Raymond, C. H., (1981) Enzyme-Linked Immunosorbent Assay for Measurement of Antibody Response to Dirofilaria immitis in Experimentally Infected Dogs. American Journal of Veterinary Research 42, 66-69. These sources and methods of generating heartworm antigens for diagnostics (from the heart of a dog) and vaccines (from mosquitos) are neither commercially feasible nor may they be socially acceptable.
I have devised a method of producing helminths (parasitic worm) antigens wherein a related species of the infectious worm that can be easily cultured is genetically modified. The method involves identifying the surface antigens of the difficult to culture parasitic worm and then creating these same antigens in the easily cultured species.
The easily cultured species is genetically altered through mutations until said species have some of the same immunological antigens of the difficult to culture parasitic worm. Through these genetic manipulations, the types and amounts of antigens of interest in the easily cultured species are altered. Antigens derived from the genetically altered species are then used for the commercial production of antigens necessary for the manufacture of diagnostics and vaccines.
The principle of my invention is to alter the antigen genes of the easily cultured species to produce preferably in situ antigens of the hard to culture parasitic species. The expression of these altered genes to produce these antigens can also occur in bacteria or yeast with the cloning of the antigen genes, and thus, provide a potentially more efficient production of these same antigens.
In the preferred embodiments, the antigens produced using this invention against the heartworm infection is by Dirofilaria immitis, and the substituted related species is the free-living hermaphroditic nematode Caenorhabditis elegans.